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Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy

Nature Protocols volume 6, pages 845–858 (2011)Cite this article

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Abstract

In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes ∼90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take ∼30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine.

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Figure 1
Figure 2
Figure 3
Figure 4: A light micrograph from a Toluidine blue–stained semi-thin section of the resin block surface.
Figure 5: Transmission electron micrographs from an unstained ultrathin section, obtained from the site of interest indicated in Figure 3, show intense contrast of cells and matrix owing to osmium ferricyanide/tannic acid treatment.
Figure 6
Figure 7
Figure 8: Final preparation of the corneal block in Quanta microscope.
Figure 9
Figure 10: Contrast reversal using ImageJ from raw FIB/SEM image of chick cornea.
Figure 11: Example sequence of contrast-reversed images selected at ten slice intervals, representative of 0.
Figure 12: The 3D reconstruction of keratocytes in ∼0.

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Acknowledgements

Our cornea research at Cardiff University is supported by project grants from the EPSRC (EP/F034970 to A.J.Q. and R.D.Y.) and Biotechnology and Biological Sciences Resource Council (BB/F022077/1 to C.K. and A.J.Q.). The use of the FEI Quanta 3D microscope at Queen Mary University of London is funded by an Engineering and Physical Sciences Research Council Access to Equipment Scheme grant (EP/F019882/1 to A.J.B.).

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Author notes

  1. Kenneth M Y P'ng

    Present address: Present address: Carl Zeiss NTS, Cambridge, UK.,

Authors and Affiliations

  1. The NanoVision Centre, School of Engineering and Materials Science, Queen Mary, University of London, London, UK

    Andrew J Bushby & Kenneth M Y P'ng

  2. Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff University, Wales, UK

    Robert D Young, Christian Pinali, Carlo Knupp & Andrew J Quantock

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  1. Andrew J Bushby
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Contributions

All authors wrote the paper. A.J.Q. designed the experimental series, of which this paper is a part, to elucidate corneal structural development. R.D.Y. carried out specimen preparation, light and transmission electron microscopy. K.M.Y.P. operated the Quanta FIB/SEM microscope, under A.J.B., supervision and collected the data sets for the 3D reconstruction. C.P. and C.K. carried out the image processing and 3D rendering.

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Correspondence to Andrew J Bushby.

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Competing interests

K.M.Y.P. is currently employed by Carl Zeiss NTS, a manufacturer of FIB/SEM instruments. K.M.Y.P. does not hold shares in Carl Zeiss NTS. The remaining authors declare that they have no competing financial interests.

Supplementary information

Supplementary Video 1

XY projection from 230 raw backscattered electron image sequence from FIB/SEM of 14 day embryonic chick cornea. (MOV 4817 kb)

Supplementary Video 2

XY projection from 230 image sequence from FIB/SEM of 14 day embryonic chick cornea after contrast reversal. (MOV 1328 kb)

Supplementary Video 3

3D reconstruction of corneal keratocytes in chick cornea at 14 day development with rotation about the y-axis showing lamellar arrangement of cells and filopodial extensions. (MOV 9045 kb)

Supplementary Video 4

3D reconstruction of corneal keratocytes in chick cornea at 14 day development with rotation about the x-axis, through an en face view of the block showing orthogonal organisation of cell filopodia through the tissue volume. (MOV 10991 kb)

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Bushby, A., P'ng, K., Young, R. et al. Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy. Nat Protoc 6, 845–858 (2011). https://doi.org/10.1038/nprot.2011.332

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